Active vitamin D3 derivatives such as 1α,25-dihydroxyvitamin D3 (hereafter “1,25 (OH)2D3 or 1,25D3) or 1α-hydroxy vitamin D3 (hereafter “1α (OH)D3) have extensive clinical applications as therapeutic substances for metabolic bone diseases such as osteoporosis. The physiological effect of these active vitamin D3 derivatives is known to be mediated by VDR. VDR is not only present in tissue such as the small intestine, bone, kidneys and parathyroid but is also present in various types of cells including immune system cells and tumor cells. Consequently active vitamin D3 derivatives are known to have various physiological effects including (1) calcium and bone metabolism regulation, (2) proliferation inhibition of tumor cells, epidermal cells and epithelial cells, and (3) regulation of immune system cells.
Animal experiments using active vitamin D3 derivatives such as 1,25 (OH)2D3 or 1α (OH)D3 have demonstrated relatively potent effects in increasing bone mass depending on the experimental conditions. On the other hand, in a clinical environment, the effect of increasing the bone density of the lumbar vertebrae or the collum femoris is poor. When using the bone density as a standard, although groups receiving doses of active vitamin D3 derivatives (clinical dosage) display superior results to groups only receiving dosages of calcium, the bone mass increase action is weaker than groups receiving administration of bisphosphonates. However, clinical results have been reported showing efficacy in suppressing fractures greater than that expected from the bone mass. The pharmacological effects described above have resulted in long-standing use as a therapeutic substance for osteoporosis. However, the mechanism for bone mass increase mediated by administration of active vitamin D3 derivatives remains unclear.
Active vitamin D3 derivatives in vitro are known to promote differentiation of osteoblasts participating in osteogenesis. However, a molecular biological mechanism of this action remains unclear.
1,25 (OH)2D3 is known to induce genetic expression of known osteoblast differentiation markers such as osteopontin and osteocalcin in bone tissue which is one of the target tissues. Osteoblasts in cell culture systems are known to differentiate as a result of 1,25 (OH)2D3 action (Non-patent Document 1). Furthermore the action of 1,25 (OH)2D3 in other target organs such as the intestines or the kidneys has long been known to induce the genes for calcium binding protein (calbindin) which participates in calcium uptake rather than to induce the genetic expression of osteoblast markers. Thus, the action depends on the type of tissue. As a result, since vitamin D3 derivatives increase blood levels of calcium, side effects such as hypercalcemia constitute problems for their clinical application. Therefore, screening for compounds lacking in, or having weak calcium blood level increase action could lead to the development of superior therapeutic substances for metabolic bone diseases with few side effects.
VDR-mediated transcriptional activity is generally known to occur through the participation of transcription co-factor (co-activator/co-repressor) in addition to VDR and the basic transcription factor. Although several transcription cofactors displaying VDR binding activity have been identified, a cofactor which may explain the role of 1,25 (OH)2D3 in the mechanism of osteoblast differentiation remains elusive. The existence of such tissue-specific genetic expression control suggests that expression or recruitment of VDR-related cofactors may depend on the tissue type. For example, differences have recently been reported in the complexes forming VDR before and after keratinocyte differentiation (Non-patent Document 2). These results suggest that cellular specificity or tissues having various intracellular genetic expression mechanisms regulated by VDR may depend on differences in the complexes forming VDR. However, identification or isolation of complexes forming VDR in osteoblasts has not been reported.
Turning now to CDP, mammalian homologues of Cut homeodomain proteins in yellow fruit flies have been isolated from mammals such as humans (Non-patent Document 3), dogs (Non-patent Document 4), mice (Non-patent Document 5) and rats (Non-patent Document 6). These homologues are respectively referred to as CDP (CCAAT Displacement Protein), Clox (Cut-like homeobox), Cux-1 (Cut homeobox) and CDP-2. Human CDP is referred to as both CUT-LIKE 1:CUTL1.
CDP belongs to a family of transcription factors present in higher order eukaryotes and is related to the control of cellular differentiation and proliferation (Non-patent Document 7). Numerous phenotypic variations in yellow fruit flies have been reported to result from insertion of a transposable insulator sequence (insulator sequence) interfering with the tissue-specific enhancer function of Cut which is a CDP yellow fruit fly homologue (Non-patent Document 8). Such phenotypes are known to be expressed in several structures in the wings (cut wings), legs, external sensory organs, Malpighian renal tubules, tracheal system or central nervous system (Non-patent Document 9). Similar to the Cux-1 and Cux-2 genes existing in mice and chickens, humans have two CDP/Cux genes: CDP-1 and CDP-2 (Non-patent Document 10). Although Cux-2 shows initial expression in nerve tissue, Cux-1 is present in almost all tissue types (Non-patent Document 10). A Cux-1 knockout mouse displays phenotypes expressed in various organs including curly whiskers, growth retardation, delayed differentiation of lung epithelia, altered hair follicle morphogenesis, male infertility, and T and B cell deficit (Non-patent Document 11). In comparison to small Cux-1 knockout mice, a genetically recombinant mouse expressing Cux-1 displays multiple organ hyperplasia and organ hypertrophy under control of the CMV enhancer/promoter. (Non-patent Document 12). In this manner, genetic experiments using yellow fruit fly and mice have shown the important role of CDP/Cux/Cut genes in the homeostasis and development of various tissues.
Expression and activation of CDP in tissue culture systems are related to cell proliferation (Non-patent Document 13), suppress CDP genetic expression occurring in terminally differentiated cells (Non-patent Document 14) and participate in the modification of matrix attachment regions (Non-patent Document 15). CDP/Cux/Cut proteins have respective DNA binding domains. All proteins contain at least one Cut homeodomain (HD) and three Cut repeats (CR1, CR2 and CR3). The cut superclass of the homeobox genes is divided into three classes: CUX, ONECUT and SATB (Non-patent Document 16). The yellow fruit fly Cut, human CDP and mouse Cux genes contain three Cut repeats and a ONECUT gene containing one Cut repeat is present in each type (Non-patent Document 10). SATB1 contains two Cut repeat-like domains and an atypical Cut-like homeodomain (Non-patent Document 17).
Although the individual Cut repeats themselves do not bind to DNA, DNA-binding affinity is enabled via certain combinations of Cut repeats or the Cut homeodomain (Non-patent Document 18). There have been two reports of intracellular DNA binding activity by CDP/Cux. CDP/Cux p200 transiently binds to DNA in the same manner as CR1CR2 and displays CCAAT substitution activity (Non-patent Document 10). In the G1/S transition of the cell cycle, proteolytic processing of the decomposable p200 protein produces CDP/Cux p110. CDP/Cux p110 contains CR2CR3HD which displays different DNA binding specificity and kinetics (Non-patent Document 19). In particular, p110 can form stable interactions with DNA. High levels of p110 isoforms are expressed in uterine leiomyomas (Non-patent Document 20).
Patent Document 1 discloses the discovery that p'75, a novel isoform of CDP/Cux, is encoded by mRNA initiated by intron 20 in the CDP/Cux site. This novel isoform displays distinct DNA binding activity to p200, p110 and p100 CDP/Cux isoforms. Although mRNA expression initiated in intron 20 is limited to certain tissue or cell types, the expression is activated in mammary tumor cell strains, primary human mammary tumors and other cancerous tissues. Consequently, antibodies against isoforms (CDP/Cux) of truncated CCAAT-substituted proteins/Cut homeobox find useful application in methods of diagnosis and prognosis for the detection of cancer. Furthermore, a method of detecting cancer by detecting RNA transcripts encoding p75 is disclosed. Moreover, CDP/Cux are known to suppress enhancer activity by competitively binding with the transcription factor binding at the matrix attachment regions of the intron enhancer (Eμ) of the immunoglobin heavy chain in a cell type specific manner or differentiation stage specific manner (Non-patent Document 21).
However these reports have no clear relationship to CDP function with respect to bone or vitamin D receptors.    Patent Document 1: International Publication No. 2004/045371 Pamphlet    Non-patent Document 1: Matsumoto et al., Bone Vol. 12, 27-32, (1991)    Non-patent Document 2: Oda A. et al., Mol. Endocrinol. Vol. 17, 2329-2339, (2003)    Non-patent Document 3: Neufeld, E. J. et al., (1992) Nat. Genet. 1, 50-55    Non-patent Document 4: Andres, V. et al., (1992) Development, 116, 321-334    Non-patent Document 5: Valarche, I. et al., (1993) Development, 119, 881-896    Non-patent Document 6: Yoon, S. O. et al., (1994) J. Biol. Chem. 269, 18453-18462    Non-patent Document 7: Nepveu (2001) Gene 270:1-15    Non-patent Document 8: Jack, et al., (1991) Development 113: 735-747    Non-patent Document 9: Jack, et al., (1991) supra    Non-patent Document 10: Neufeld, E. J., et al., (1992) supra    Non-patent Document 11: Ellis, et al., (2001) supra    Non-patent Document 12: Ledford, et al., (2002) Dev. Biol. 245: 157-171    Non-patent Document 13: Holthuis, et al., (1990) Science 247: 1454-1457    Non-patent Document 14: Pattison, et al., (1997) J. Virol. 71: 2013-2022    Non-patent Document 15: Liu, et al., (1997) Mol. Cell. Biol. 17:5275-5287    Non-patent Document 16: Burglin and Cassata (2002) Int. J. Dev. Biol. 46:115-123    Non-patent Document 17: Dickinson, et al. (1997) J. Biol. Chem. 272: 11463-11470    Non-patent Document 18: Moon, et al., (2000) J. Biol. Chem. 275: 31325-31334    Non-patent Document 19: Moon, et al., (2001) Mol. Cell. Biol. 21: 6332-6345    Non-patent Document 20: Moon, et al., (2001) supra    Non-patent Document 21: Wang, et al., (1999) MOLECULAR AND CELLULAR BIOLOGY, 19:284-295